We will continue to elucidate the subcellular physiology of intraerythrocytic P. falciparum malarial parasites using established and newly developed advanced cellular imaging techniques, and genetically well defined strains of parasites. As recent work from this laboratory and others have shown, differences in this subcellular physiology exist for chloroquine resistant (CQR) vs. sensitive (CQS) malarial parasites. These differences help define the molecular basis of drug resistance in malarial parasites and thus aid the design of new antimalarial chemotherapy. Specifically, we will: Further molecularly define intracellular pH regulation for drug resistant malarial parasites in vivo;(Aim 1) Define additional physiologic parameters associated with malarial parasite drug resistance;(Aim 2) Determine the relative contribution of Pfcrt, Pfmdr1, and newly described QN resistance loci to these alterations in parasite physiology (Aim 3). The proposed work is interdisciplinary and innovative. We will use novel cell culture techniques, genetically modified P. falciparum, our established single cell photometry (SCP) and laser confocal microscopy techniques, and, for the first time to our knowledge, novel spinning disk confocal microscopy (SDCM) methods. These SDCM methods allow us to acquire complete, 3 dimensional images of living intraerythrocytic parasites under constant perfusion, and at submicron resolution, within a remarkable 1 second (or less). Thus, answers to key questions that rely on (for example) rapidly quantifying sub cellular organellar volume at very high spatial resolution, are now attainable. Knowledge gained during this research will significantly expand our understanding of malarial parasite physiology and antimalarial drug resistance.